The objectives of this proposal are to determine the structure, function and catalytic mechanisms of several enzyme families involved in yeast/fungal glycoprotein biosynthesis and to study the role of the yeast ER alpha1,2-mannosidase, Mnsl p, and its enzymatically inactive homolog, Htmlp, in quality control. The first family consists of Class I alpha1,2-mannosidases that have been conserved in all eukaryotes. The ER members of this family participate in endoplasmic reticulum quality control, particularly in the degradation of misfolded glycoproteins, a process known as ERAD. The Golgi members are essential for the formation of complex N-glycans in mammalian cells. They are potential targets for the development of antimetastatic, antiviral agents, and for the development of inhibitors that would stabilize misfolded glycoproteins in genetic diseases characterized by rapid degradation of newly formed glycoproteins. We have determined the three dimensional structure of several Class I alpha1,2-mannosidases by X-ray crystallography. We plan additional X-ray crystallographic studies on wild type and mutant enzyme to elucidate the catalytic mechanism of the Class I alpha1,2-mannosidase family. We plan to express recombinant Html p to determine its structure and its recognition properties for misfolded glycoproteins. The other enzyme families are yeast/fungal-specific Golgi alpha-mannosyltransferases required for cell wall synthesis. These enzymes are potential targets for the development of antifungal agents. We have determined the structure of the first yeast Golgi alphamannosyltransferase, Kre2p, and plan to elucidate its catalytic mechanism. We also plan structure-function studies of Golgi alpha1,6-mannosyltransferases required for cell wall mannan synthesis. In addition, we have shown that the yeast membrane protein split ubiquitin system is useful to demonstrate membrane protein interactions. We plan to screen split ubiquitin membrane yeast two hybrid libraries to identify novel membrane proteins involved in targeting glycoproteins for ERAD.